RNeasy Protect Bacteria Mini Kit

For stabilization and purification of up to 100 µg total RNA from bacteria

Immediate in vivo RNA stabilization and protection

Reliable gene expression and gene-profiling data

No need for liquid nitrogen, dry ice, or phenol

Ready-to-use, high-quality total RNA in minutes

No CsCl gradients, no LiCl or ethanol precipitation

The RNeasy Protect Bacteria Mini Kit includes RNAprotect Bacteria Reagent for stabilizing RNA in bacterial samples, and RNeasy spin columns for purifying up to 100 µg of high-quality RNA using silica-membrane technology. Efficient disruption of bacterial samples can be achieved using the TissueLyser II. The kit can be automated on the QIAcube.

The RNeasy Protect Bacteria Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Accurate gene expression profiles.

Total RNA was isolated from RNAprotect stabilized E. coli cultures using the RNeasy Protect Bacteria Kit (RNeasy Protect Bacteria) or from unstabilized cultures using a commercial precipitation method (Supplier EIII). To distinguish between gene expression under defined culture conditions and effects of artifactual gene induction during harvesting and RNA isolation, the RNA polymerase inhibitor rifampicin was added to half of the culture prior to RNA isolation. Differences in transcript levels with and without rifampicin therefore generally reflect the degree of RNA degradation. [A]GeneChip analysis of E.coli microarrays was carried out according to standard Affymetrix protocols. [B] The percentage of genes expressed was estimated as the number of different transcripts determined present by GeneChip analysis divided by the total number of transcripts represented on the microarray. (Data from a collaborative study with Affymetrix.)

RNAprotect Bacteria Reagent procedure.

RNAprotect Bacteria Reagent prevents mRNA degradation.

In order to monitor mRNA degradation only, transcription was stopped by adding the RNA polymerase inhibitor rifampicin to a growing culture of E. coli. The culture was split into halves, and RNAprotect Bacteria Reagent was added to one half. Samples were left at room temperature for 0, 4, 8, and 16 minutes before centrifugation and RNA isolation. The resulting RNA was analyzed by agarose gel electrophoresis (top panel). The expression of two marker genes with different half-lives was examined by northern blot analysis. Middle panel: ompA (half life of 15 minutes); bottom panel: α-lactamase (half life of 2–5 minutes).

During traditional methods for cell harvesting and RNA isolation, enzymatic degradation of RNA leads to reduction or loss of many transcripts. The reduction is particularly significant in bacterial mRNA molecules, which have very short half lives of only a few minutes. In addition, genes can be induced during handling and processing of bacterial samples, leading to higher expression. Use of RNAprotect Bacteria Reagent overcomes these problems by providing immediate stabilization prior to RNA isolation (see figures "RNAprotect Bacteria Reagent prevents mRNA degradation" and "Accurate gene expression profiles"). Yield from E. coli (6 x 108 cells) is 100 µg of RNA and the RNA yield from Bacillus subtilis (1 x 108 cells) is 15 µg. RNA purified with the kit is high-quality with A260/A280 ratios of 1.9–2.1 (measured in 10 mM Tris·Cl, pH 7.5).

Principle

The RNeasy Protect Bacteria Mini Kit provides a complete RNA protection and isolation solution, from sample harvesting to pure RNA, in one kit. Proven RNeasy technology, combined with the RNA-stabilizing properties of RNAprotect Bacteria Reagent, allows purification of high-quality, intact RNA. This process efficiently preserves the expression profile of the bacteria at the time of harvesting to ensure reliable gene expression analysis. Following stabilization, RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification (see figure "RNeasy Mini spin column").

Procedure

Two volumes of RNAprotect Bacteria Reagent are added directly to 1 volume of bacterial culture (≤7.5 x 108 bacteria) prior to RNA isolation, providing immediate stabilization of RNA (see flowchart "RNAprotect Bacteria Reagent procedure"). The stabilization allows time for efficient bacterial lysis using a choice of protocols: enzymatic lysis, mechanical disruption, or a combination of both methods. We recommend the TissueLyser II for efficient mechanical disruption. Ethanol is then added to the lysate to provide ideal binding conditions. The lysate is loaded onto the RNeasy silica membrane (binding capacity 100 µg RNA). Following RNA binding, all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in 30–100 µl of water. The RNeasy Protect Bacteria Mini Kit can be automated on the QIAcube.

Amounts of RNA isolated from bacteria can vary due to species and growth conditions. The RNeasy Protect Bacteria Mini Kit is suitable for use with a wide range of bacterial species, both Gram positive (e.g., Staphylococcus aureusand Mycobacterium avium) and Gram negative (e.g., Escherichia coliand Salmonella typhimurium). Bacteria grown in either minimal or complex medium can be used. Since the RNeasy procedure enriches for RNA species >200 nucleotides, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs. RNeasy Protect Bacteria Kits provide the highest-quality RNA with minimum copurification of DNA. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using the RNase-Free DNase Set for convenient on-column DNase treatment during the RNeasy procedure.

Applications

RNA purified with the RNeasy Protect Bacteria Mini Kit is ideal for use in all applications. Downstream applications include: